Project Summary Fragile X mental retardation syndrome is the most common form of inherited mental retardation, affecting ~ 1 in 3600 males and ~ 1 in 6000 females. The syndrome is caused by the loss of a normal cellular protein, named the fragile X mental retardation protein (FMRP). Despite extensive research in the past two decades, the relationship between the absence of FMRP and the phenotype of the fragile X syndrome is still not fully understood. FMRP is an RNA binding protein involved in the transport and translation regulation of specific messenger RNA (mRNA) targets. Biochemical studies have determined that FMRP uses its arginine-glycine- glycine (RGG) box to bind with high affinity to RNA sequences that form G quadruplex (GQ) structures. The mechanisms by which FMRP exerts its translation regulator function are not known, however it has been proposed that the protein works in conjunction with the microRNA (miRNA) pathway to regulate local protein synthesis in response to synaptic input. This proposal has the following specific aims: 1. Investigation of the FMRP role in miRNA maturation. Several miRNAs have been shown to require FMRP to exert their function, and FMRP has also been shown to bind pre-miRNAs. We hypothesize that FMRP regulates the maturation of specific miRNAs through its interactions with GQ structures formed by their precursor pre-miRNAs. To test this hypothesis we will characterize the GQ structures predicted to form in selected pre-miRNAs and analyze their interactions with FMRP. Subsequently, we will analyze the effect of FMRP upon the production of mature miRNAs by Dicer. 2. Investigation of FMRP: GQ mRNA: miRNA interactions in translation regulation. FMRP has been shown to directly regulate the translation of specific mRNAs through its interactions with the miRNA-guided RNA induced silencing complex (RISC). Following the guiding hypothesis that FMRP interacts with GQ structures in specific mRNAs to modulate their recognition by RISC, under this aim we will analyze FMRP: GQ mRNA: miRNA interactions in the context of other mRNAs that have predicted GQ structures: (i) within their miRNA binding sites and (ii) immediately adjacent to them. 3. Investigation of various FMRP isoforms interactions with the miRNA pathway. We hypothesize that the FMRP isoforms created by alternative splicing at exon 12 will have different interactions with miRNA precursors and/or protein components of the miRNA pathway, which could potentially result in functional differences with respect to translation regulation. Additionally, we hypothesize that FMRP activity-dependent primary and secondary phosphorylation events could further modulate these functional differences.